Gram-negative bacterial pneumonia confers a high rate of morbidity and mortality. Klebsiella pneumonia (KP) is a common cause of both community acquired and nosocomial pneumonia and is responsible for a growing number of antibiotic-resistant infections. Increasing rates of antibiotics resistance highlight the importanc of understanding innate immune responses to infection, in order to potentially exploit these mechanisms clinically. The IL-36 family is a group of novel IL-1-like pro- inflammatory cytokines that are highly expressed in skin and epithelial tissues, as well as some bone marrow- derived cell types. IL-36g induces neutrophilic inflammation and induction of inflammatory cytokines and chemokines in a murine model of asthma, however little is known regarding its role in lung infections. In our preliminary studies, we have found that IL-36g is produced in the lung in response to (KP) infection. In vivo, we have seen that IL-36g blockade results in the impairment of bacterial clearance as well as enhancement of bacterial dissemination in a mouse model of KP pneumonia. This may be due at least in part to IL-36- mediated Th1-driven responses. Mechanisms of secretion of IL-36 family members are not well understood. We have seen that although cells will secrete some IL-36g in response to KP, ATP is required for optimal secretion, just as with IL-1b. Furthermore, our preliminary studies suggest that IL-36g may be secreted in microparticles for delivery to target cells. These preliminary studies provide the rational for our hypothesis that IL-36 family members are secreted via microparticles in response to infection, and are important components of effective innate immunity against Gram-negative pneumonia. Specific Aims. To test our hypothesis, we propose to 1) determine the cellular sources and mechanisms of secretion of IL-36g in the lung in response to Gram-negative infection, 2) determine the effect of IL-36g on host immune responses in vivo and in vitro, and 3) examine the interaction of IL-36g with dendritic cells to promote a Th1 response. Experimental Approach. WT and either IL-36g or IL-36R-deficient mice (through genetic deletion or antibody neutralization) were exposed to intratracheal KP inoculation, and bacterial counts from lungs, blood, and spleen were assessed. Survival and alveolar leukocyte influx will also be assessed in response to in vivo infection. Alveolar epithelial cells, lung macrophages, and bone marrow derived dendritic cells were isolated from WT mice and treated with live or heat-killed KP to assess IL-36g induction. Additionally, cells were treated with IL-36g to assess for chemokine/cytokine induction. Significance of the Results. Our preliminary data shows that IL-36g is induced in response to a variety of exogenous pathogen associated molecular patterns and live bacteria, and that it is secreted in response to KP in vitro. We have also shown that IL-36 blockade during infection results in reduced alveolar macrophage and neutrophil influx during experimental Klebsiella pneumonia, resulting in decreased bacterial clearance and enhanced dissemination. Taken together, these studies suggest that IL-36g may act as an alarmin, being produced and released as a consequence of infection as an endogenous marker of cell injury/death.